Gene therapies for neurodegenerative disorders targeting ganglioside biosynthetic pathways

ABSTRACT

A composition of matter comprising an adeno-associated virus (AAV) or other human compatible virus, encoding the gene for Sialidase Neu3, B3Galt4, St3Gal2, or combinations thereof, and a neuron specific promoter, wherein the composition is suitable for administration to a patient comprising injecting the AAV or other human compatible virus into the brain by intracranial stereotaxic injunction; wherein the AAV&#39;s encoding for the Sialidase Neu3, B3Galt4, St3Gal2, or combinations thereof enhance and/or normalize levels of GM1 in neurons, providing both therapeutic relief and disease modifying effects in specific areas of the brain relevant to particular neurodegenerative diseases.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 62/194,910, filed Jul. 21, 2015 and 62/194,954, filed Jul. 21, 2015,the disclosure contents of which are hereby incorporated by reference intheir entirety.

FIELD OF INVENTION

The present application is generally related to therapeutic treatmentsfor neurodegerative disorders including Parkinson's disease (PD) andHuntington's disease (HD) comprising administration of a Sialidase Neu3through gene therapy, through administration of B3Galt4 cDNA or acombination thereof, to a patient suffering from a neurodegenerativedisease.

BACKGROUND OF THE INVENTION

HD is a hereditary autosomal-dominant, progressive neurodegenerativedisorder, resulting from expansion of a polygultamine (CAG) repeat inthe gene coding for the Huntingtin (Htt) protein, leading to formationof mutant Htt (mHtt), which confers a toxic gain of function, withneurons of the striatum being particularly vulnerable. Accumulation ofmHtt drives neuronal dysfunction via transcriptional dysregulation,modification of cell signaling, abnormal axonal transport and synapticactivity leading to clinical signs and symptoms including chorea,dystonia, incoordination, cognitive decline and behavioral difficulties,culminating with death. Latency from diagnosis to death is typicallyapproximately 20 years. Death is commonly due to complications offalling and/or dysphagia and aspiration, although it is estimatedthat >25% of patients attempt suicide at some point after diagnosis.

While genetic testing is available for HD, less than 5% of at-riskindividuals elect to have predictive testing, with many citing the lackof an effective treatment as the rational for this decision. Currentlythe standard of care for HD is symptomatic, utilizing modestly effectiveanti-choreic drugs such as tetrabenazine. The use of symptomatic therapydoes not alter the course of the disease. Survival rates in treated v.untreated patients are similar. As such, there is a significant unmetmedical need for an effective, disease modifying therapy for HD.

Recent research found that as well as the mHtt toxicity in striatal (andcortical) neurons in HD, there is a significant dysregulation ofganglioside biosynthesis in these cells in HD models. Similar defectswere found in fibroblasts from HD patients, suggesting thatdysregulation in ganglioside expression may be a universal phenomenon inHD. Gangliosides, sialic acid containing sphingolipids, are majorcomponents of membrane lipid rafts where they act as modulators of cellsignaling. Found throughout the body, gangliosides are highly enrichedin the central nervous system (CNS), where they are critical to normalCNS development and neuron survival. Maglione et al. described impairedganglioside metabolism in tissues from HD patients (fibroblasts andbrain) and in a transgenic model of HD in mice (YAC128). These findingshave been confirmed by others and extended to the R6/1 transgenic HDmouse, further strengthening the correlation between gangliosidedysregulation and HD. Primarily, the monosialoganglioside (GM1) issignificantly reduced in both HD and PDmodels, although downstreampolysialogangliosides such as GD1a, GD1b, GT1b are also partiallyaffected. The administration of bovine brain-derived GM1 by infusionpump to YAC128 mice that were symptomatic for motor impairment resultedin normalization of motor function within 2 weeks of the start oftreatment and lasting until the last day of infusion and resulted inphosphorylation of mHtt at serine residues 13 and 16 that attenuatedmHtt toxicity, suggesting a disease modifying effect of GM1administration.

Parkinson's disease is another neurodegenerative disorder that slowlyprogresses and is characterized by loss of dopamine-producing neurons inthe substantia nigra, loss of forebrain dopamine, and a time-dependentworsening of clinical symptoms. Although symptomatic improvement can beobtained with available pharmacotherapies, functional abilitydeteriorates over time with the progressive loss of nigrostriataldopamine neurons. Development of therapies that can slow the progressionof the disease would fill a major unmet medical need in PD.

It is estimated that PD is the second most common neurodegenerativedisease of mid to late life, with estimates of 1 of every 200 persons inthe US aged 60-69 having PD. Additionally, 1 in 100 persons over 70 and1 in every 35 persons over 80 suffer from PD. Worldwide, numbers suggestthat between 4 to 5 million people suffer from PD and that number coulddouble to between 8-10 million by 2030.

Current gold-standard treatment for PD is a drug first discovered in the1960's, levodopa, which strictly provides symptomatic relief. Newtherapies are needed to provide symptomatic relief and protectionagainst continued neuronal degeneration to those who suffer fromneurodegenerative diseases such as PD.

As in HD, there are decreased levels of GM1 in the PD brain(particularly in the substantia nigra) and administration of bovinebrain-derived GM1 is neuroprotective in animal and cell models of PD andhas both symptomatic effects and slows disease progression in PDpatients. However, due to drawbacks associated with the administrationof animal brain-derived GM1, including the low ability of systemicallyadministered GM1 to gain access to the brain, there is a need for newmethods of treatment for increasing GM1 levels in the brain.

SUMMARY OF THE INVENTION

In accordance with these and other objects, a first embodiment of aninvention disclosed herein is directed to a composition of mattercomprising a viral vector expressing an exogenous nucleic acid sequencecomprising a functional sialidase Neu3 enzyme and a promoter operablylinked to said nucleic acid sequence. Said composition can suitably beutilized for treatment of neurodegenerative diseases or disorders tomodify the amounts of GM1 in the brain.

A method of increasing the amount of GM1 in a brain tissue comprisingadministering to a patient an effective amount of an AAV encoding forsialidase Neu3, to the patient, wherein the sialidase Neu3 is effectivein increasing the amounts of GM1 in the brain tissue of interest.

A method of increasing the amount of GM1 in a brain tissue comprising aconcomitant treatment of GM1 and gene therapy comprising an AAV encodingfor sialidase Neu3, wherein the GM1 is administered to directly increaseGM1 levels in the brain and the AAV is administered to upregulate theproduction of GM1 through modification of the sialidase Neu3 so as tomodify and stabilize the levels of GM1 in the brain; and wherein thelevels of GM1 in the brain are thereafter increased as compared to thelevels prior to administration.

A further embodiment is directed to a method of increasing the amount ofGM1 in a brain tissue comprising a first step of administering aneffective amount of an AAV encoding for sialidase Neu3 to a patientfollowed by a concomitant therapy comprising administration of an AAV toadminister a gene encoding for B3Galt4. Based on experimental data, thegene encoding St3Gal2 (which converts GM1 to GD1a and GD1b to GT1b) isalso downregulated in HD models and HD fibroblasts. Furthermore, thereis some evidence that St3Gal2 is also downregulated in PD. Therefore, anappropriate AAV to administer St3Gal2 can also be utilized to increaselevels of GD1a and GT1b which could then be converted into GM1 via AAVNeu3.

A further embodiment is directed to a method for treating aneurodegenerative disease or condition comprising administering aneffective amount of a human compatible engineered adeno-associated virus(AAV) or lentivirus containing the B3Galt4 sequence under the control ofneuronal specific promoters administered to the patient with aneurodegenerative disease or condition by intracranial stereotaxicinjunction or by systemic administration.

A further embodiment is directed to a method to increase endogenous GM1ganglioside in the substantia nigra in a PD patient or in the caudatenucleus, the putamen and/or additional affected brain regions in an HDpatient, by administering an effective amount of a composition encodingfor Neu3, and optional concomitant administration of shRNA or siRNAdirected against St3gal2 to increase GM1 levels. Through administrationof a Neu3-based therapy, it would be an advantage to try to increaseGD1a and GT1b which are substrates for Neu3 and will help to allow moreGM1 to be made.

A further embodiment is directed to a method for increasing GM1 levelsin a patient suffering from a neurodegenerative disease or conditioncomprising administering to said patient an effective amount of an shRNAor siRNA for St3gal2, sufficient to increase GM1 levels in said patientwith a concomitant therapy comprising administration of B3Galt4 cDNA tosaid patient.

A further embodiment is directed to a method to increase endogenous GM1ganglioside in the brain comprising administering GM1 directly to apatient suffering from a neurodegenerative disease and administeringB3Galt4 cDNA to increase production of GM1 in the brain; wherein thedirect administration provides direct increase of GM1 in the brain andthe B3Galt4 cDNA provides protective and restorative effects bymodulating and increasing the native production of GM1 in the brain.

A further embodiment is directed to a method to increase endogenous GM1ganglioside in the brain comprising administering GM1 directly to apatient suffering from a neurodegenerative disease or condition andadministering B3Galt4 cDNA and concomitantly administering shRNA/siRNASt3gal2 to increase levels of GM1 in the brain and decrease degradationof GM1; wherein the direct administration of GM1 increases GM1 directlyand the B3Galt4 cDNA and shRNA/siRNA for St3gal2 provide protective andrestorative effects by modulating and increasing the native productionof GM1 in the brain.

A further embodiment is a method of increasing the amount of GM1 in abrain tissue comprising a concomitant treatment of a dietary supplementwith complex milk lipids (CMLs) having a concentrated dietary source ofGM3, to provide increase levels of substrate GM3 and GM2 for conversionto GM1 and gene therapy comprising an AAV or lentivirus encoding forB3Galt4, wherein the CMLs provide increased substrate to convert GM3 andGM2 to GM1 and increased expression of B3Galt4 increases conversion ofGM2 to GM1 in the brain. The AAV or lentivirus or non-viral vector isadministered to up regulate the production of B3Galt4; and wherein thelevels of GM1 in the brain are thereafter increased as compared to thelevels prior to administration.

A further embodiment is directed to a composition of matter suitable forincreasing GM1 in the brain comprising a vector expressing an exogenousnucleic acid sequence comprising a functional sialidase directed to aNeu3 enzyme, B3Galt4, St3Gal2, or combinations thereof, and a promoteroperably linked to said nucleic acid sequence. Said composition cansuitably be utilized for treatment of neurodegenerative diseases ordisorders to modify the amounts of GM1 in the brain.

A further embodiment is directed to a method for treating aneurodegenerative disease or condition comprising administering aneffective amount of a human compatible engineered non-viral vectorsincluding liposomes, polymersomes, lipopolyplex, or bolaamphiphilenanovesicles (that can be targeted to specific neuron subtypes)containing the Neu3 sequence under the control of neuronal specificpromoters. The same basic strategies for non-viral vectors are alsosuitably used for B3Galt4 or St3Gal2 as described herein.

A further method is directed to treating a neurodegenerative diseasecomprising administering to a patient a human compatible lentivirusencoding for Sialidase Neu3, B3Galt4, or both. Appropriate strategiesmay employ use of any one of the compositions of matter as a medicamentfor treating a neurodegenerative disorder or use of said compositions ofmatter for increasing GM1 in the brain.

A method of increasing the amount of GM1 in a brain tissue comprising aconcomitant treatment of a dietary supplement with complex milk lipids(CMLs) having a concentrated dietary source of GM3, to provide increaselevels of substrate GM3 and GM2 for conversion to GM1 and gene therapycomprising an AAV encoding for sialidase Neu3, wherein the CMLs provideincreased substrate to convert GM3 and GM2 to GM1 in the brain and theAAV is administered to up regulate the production of GM1 throughmodification of sialidase Neu3 so as to modify and stabilize the levelsof GM1 in the brain; and wherein the levels of GM1 in the brain arethereafter increased as compared to the levels prior to administration.

A composition of matter comprising an adeno-associated virus (AAV) orother human compatible virus, encoding the gene for Sialidase Neu3,B3Galt4, St3Gal2, or combinations thereof, and a neuron specificpromoter, wherein the composition is suitable for administration to apatient comprising injecting the AAV or other human compatible virusinto the brain by intracranial stereotaxic injunction; wherein the AAV'sencoding for the Sialidase Neu3, B3Galt4, St3Gal2, or combinationsthereof enhance and/or normalize levels of GM1 in neurons, providingboth therapeutic relief and disease modifying effects in specific areasof the brain relevant to particular neurodegenerative diseases.

Therefore, there is a need for new methods of treatment ofneurodegenerative diseases such as PD and HD, including treatments andmechanisms for regulating or reversing a defect in B3Galt4 expressionthrough administration of B3Galt4 cDNA or for administering a sialidaseNeu3 cDNA, or combinations thereof, to increase GM1 in the brain.

Additional features and embodiments will be apparent to one of ordinaryskill in the art upon consideration of the following detaileddescription of preferred embodiments and descriptions of the best modeof carrying out the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B depicts a flowchart showing ganglioside biosyntheticpathways.

FIG. 2 depicts an experimental overview of hES cells treated with MPP⁺and GM1 or sialidase.

FIGS. 3A and 3B depict the protection/rescue of human embryonic stemcells from MPP⁺ toxicity.

FIG. 4 depicts striatal uptake of tritiated dopamine (³H-DA).

FIG. 5 depicts immunofluorescent detection of gangliosides in mousebrain and effects of sialidase on dopamine levels and number of dopamineneurons.

FIGS. 6A and 6B depict experimental overviews of certain mouse studies.

FIG. 7 depicts the effects of sialidase treatment on striatal dopaminelevels in a PD model.

FIGS. 8A, 8B, 8C, and 8D depict the effects of sialidase treatment onsubstantia nigra neurons in a PD model.

FIG. 9 depicts AAV-hNeu3 expression in mouse substantia nigra 2 weeksafter administration.

FIG. 10 depicts neuroprotection in an MPTP mouse PD model with AAV-hNeu3administration.

FIG. 11 depicts symptomatic and disease modifying effects of GM1 in PDpatients.

FIG. 12 depicts that some gangliosides are reduced in PD substantianigra.

FIG. 13A-B depicts changes in B3Galt4 gene expression SN and decreasedB3Galt4 protein expression in human PD substantia nigra.

FIG. 14A-B depicts enhancement of cell death when B3Galt4 isdownregulated.

FIG. 15A-B depicts downregulation of B3Galt4 in HD models and HDfibroblasts.

FIG. 16 depicts neuroprotective effect of AAV-B3Galt4 in the MPTP mousePD model.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The embodiments of the invention and the various features and advantagesthereto are more fully explained with references to the non-limitingembodiments and examples that are described and set forth in thefollowing descriptions of those examples. Descriptions of well-knowncomponents and techniques may be omitted to avoid obscuring theinvention. The examples used herein are intended merely to facilitate anunderstanding of ways in which the invention may be practiced and tofurther enable those skilled in the art to practice the invention.Accordingly, the examples and embodiments set forth herein should not beconstrued as limiting the scope of the invention, which is defined bythe appended claims.

As used herein, terms such as “a,” “an,” and “the” include singular andplural referents unless the context clearly demands otherwise.

The term “increase” is generally used to describe a positive change inthe levels of GM1 as compared to pre-treatment levels in the brain.

The term “AAV” means adeno-associated virus.

The term “B3Galt4” means beta-1,3-galactosyltransferase 4 or GM1synthase.

The term “Sialidase Neu3” means neuraminidase enzyme Neu3.

As used herein, the term “about” means plus or minus 5% of the numericalvalue of the number with which it is being used. Therefore, about 50%means in the range of 45%-55%.

“Administering” when used in conjunction with a therapeutic means toadminister a therapeutic directly to a subject, whereby the agentpositively impacts the target. “Administering” a composition may beaccomplished by, for example, injection, oral administration, topicaladministration, or by these methods in combination with other knowntechniques. Such combination techniques include heating, radiation,ultrasound and the use of delivery agents. When a compound is providedin combination with one or more other active agents (e.g. otheranti-atherosclerotic agents such as the class of statins),“administration” and its variants are each understood to includeconcurrent and sequential provision of the compound or salt and otheragents.

By “pharmaceutically acceptable” it is meant the carrier, diluent,adjuvant, or excipient must be compatible with the other ingredients ofthe formulation and not deleterious to the recipient thereof.

“Composition” as used herein is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationof the specified ingredients in the specified amounts. Such term inrelation to “pharmaceutical composition” is intended to encompass aproduct comprising the active ingredient(s), and the inert ingredient(s)that make up the carrier, as well as any product which results, directlyor indirectly, from combination, complexation or aggregation of any twoor more of the ingredients, or from dissociation of one or more of theingredients, or from other types of reactions or interactions of one ormore of the ingredients. Accordingly, the pharmaceutical compositions ofthe present invention encompass any composition made by admixing theviral or non-viral vectors, cDNA or other delivery vehicles described inthe present invention and a pharmaceutically acceptable carrier.

As used herein, the term “agent,” “active agent,” “therapeutic agent,”or “therapeutic” means a compound, composition, or viral or non-viraldelivery vehicle utilized to treat, combat, ameliorate, prevent orimprove an unwanted condition or disease of a patient. Furthermore, theterm “agent,” “active agent,” “therapeutic agent,” or “therapeutic”encompasses a combination of one or more of the compounds of the presentinvention.

A “therapeutically effective amount” or “effective amount” of acomposition is a predetermined amount calculated to achieve the desiredeffect, i.e., to inhibit, block, or reverse the activation, migration,proliferation, alteration of cellular function, and to preserve thenormal function of cells. The activity contemplated by the methodsdescribed herein includes both medical therapeutic and/or prophylactictreatment, as appropriate, and the compositions of the invention may beused to provide improvement in any of the conditions described. It isalso contemplated that the compositions described herein may beadministered to healthy subjects or individuals not exhibiting symptomsbut who may be at risk of developing a particular disorder. The specificdose of a compound administered according to this invention to obtaintherapeutic and/or prophylactic effects will, of course, be determinedby the particular circumstances surrounding the case, including, forexample, the compound administered, the route of administration, and thecondition being treated. However, it will be understood that the chosendosage ranges are not intended to limit the scope of the invention inany way. A therapeutically effective amount of composition of thisinvention is typically an amount such that when it is administered in aphysiologically tolerable excipient composition, it is sufficient toachieve an effective systemic concentration or local concentration inthe tissue depending on the form of administration.

The terms “treat,” “treated,” or “treating” as used herein refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological condition, disorder, or disease, or to obtain beneficialor desired clinical results. For the purposes of this invention,beneficial or desired results include, but are not limited to,alleviation of symptoms; diminishment of the extent of the condition,disorder, or disease; stabilization (i.e., not worsening) of the stateof the condition, disorder, or disease; delay in onset or slowing of theprogression of the condition, disorder, or disease; amelioration of thecondition, disorder, or disease state; and remission (whether partial ortotal), whether detectable or undetectable, or enhancement orimprovement of the condition, disorder, or disease. Treatment includesprolonging survival as compared to expected survival if not receivingtreatment.

In various animal models of PD, systemic administration of the a-seriesganglioside, GM1, protects dopaminergic (DAergic) neurons fromdegeneration and restores function to damaged neurons. In a recentclinical trial, administration of GM1 to PD patients resulted insymptomatic improvement and slowed the progression of symptoms.Ganglioside deficiencies and imbalances have been observed in aging andhave been suggested to occur in neurodegenerative diseases and thesechanges may exist in the PD brain. There may be a specific GM1deficiency and possible decrease in other gangliosides in PD thatcontributes to neurodegeneration of the nigrostriatal pathway. Toexplore this possibility, we examined the expression of GM1 (as well asother a- (ex., GD1a) and b- (ex., GD1b and GT1b) series gangliosides) aswell as mRNA expression for some of enzymes responsible for gangliosidebiosynthesis in the PD brain. We have also examined expression ofganglioside-related genes in dopamine neurons isolated from post mortemPD brain and normal control brain.

The more highly expressed gangliosides in the brain are GM1, GD1a, GD1b,GT1b, and GD3. GD3 is a potential mediator of apoptosis whereas GM1 hasbeen shown to be broadly neuroprotective in a variety of systems andmodels. Preclinical studies showed that GM1 treatment in various PDmodels resulted in significant behavioral and biochemical recoveryincluding protection/rescue of damaged dopamine (DA) neurons andincreased DA levels in the striatum. A recent randomized, controlled,delayed start clinical study showed that in PD patients, GM1 use for thefirst 24 weeks of the study was superior to placebo for improving motorsymptoms and that extended GM1 use (up to 120 weeks) resulted in asustained clinical benefit and lower than expected symptom progression.These data suggest that enhancing GM1 levels in PD may be diseasemodifying. Although GM1 administration has promise as a treatment forPD, it is difficult to source, is currently available only as a naturalproduct extracted and purified from animal brains, and has low abilityto cross the blood brain barrier.

The reasons why GM1 treatment is so effective clinically in PD are notcompletely clear but it is possible that PD is characterized in part bya disorder of glycolipid biosynthesis/metabolism, in particular, a GM1deficiency disorder, and that GM1 administration represents GM1replacement therapy. The pathophysiology of the disease may involvealterations in the pathways involved with ganglioside biosynthesisand/or metabolism in the PD brain.

An alternative therapeutic approach to administering GM1 is to increaseendogenous GM1 levels in the brain. One method for enhancing GM1 levelsin the brain involves the manipulation of ganglioside degradation bysialidases. Sialidases hydrolyze sialic acid linkages on gangliosidesand can degrade complex gangliosides (and GD3) while increasing GM1(where the sialic acid linkage is not susceptible to the sialidase).Specifically, sialidases catalyze the removal of α-linked sialic acidresidues from carbohydrate groups of gangliosides. Thus, gangliosidesGD1b, GT1b, and GD1a can be converted to GM1 and the potentiallyapoptogenic GD3 can be degraded.

The human sialidase Neu3 has unique substrate specificity (i.e.,degrades complex gangliosides GD1a, GT1b, and GD1b into GM1, does notcleave internal sialic acids on GM1 and GM2, and hydrolyzes GD3) andassociates with plasma membranes. Neu3 is active toward plasma membranegangliosides where it can modify the ganglioside content, specificallyincreasing GM1 levels and potentially modifying important functionalmembrane events such as cell signaling. Neu3 also possessesanti-apoptotic properties. Transfection of the Neu3 gene into cancercells up-regulated Neu3 and inhibited apoptosis, increased Bcl-2protein, and decreased caspase-3 expression. In contrast, silencing Neu3expression induced apoptosis without specific stimuli and wasaccompanied by decreased Bcl-xL (pro-survival protein) expression. Ourstudies have shown that this process can be stimulated in vivo and itconfers neuroprotection in PD models similar to that achieved withadministration of GM1.

Indeed, the most highly expressed gangliosides in brain are GM1, GD1a,GD1b, GT1b, and GD3. While GM1 is broadly neuroprotective in a varietyof systems and models, GD3 is a potential mediator of apoptosis. Thetherapeutic method described in this invention will simultaneouslydecrease GD3 levels (a potential mediator of toxicity) and/or increaseGM1 levels (potentially neuroprotective), providing a superiorneuroprotective effect. However, any increase in GM1 levels, evenwithout concomitant depreciations in GD3 levels, provides aneuroprotective effect.

In HD models, it has been shown that intracranial administration ofbovine brain-derived GM1 resulted in normalized motor function, aconcomitant normalization of striatal dopamine levels (critical tonormal striatal function), and phosphorylation of mHtt at serineresidues 13 and 16 that attenuated mHtt toxicity. These data suggestthat enhancing GM1 levels in HD may also be disease modifying.

Therefore, because each of HD and PD exhibit decreases in GM1ganglioside levels, mechanisms to support GM1 levels or increase GM1levels will provide protective and stabilizing effects in the brain.

Unfortunately, no drug has yet been developed that has an unequivocalpositive effect on disease progression. What is needed is a newtreatment for PD that slows the degeneration of substantia nigradopamine neurons and thus slows, arrests, or reverses the progression ofthe disease.

GM1 levels can be increased in the brain in several different ways. Afirst mechanism is to simply administer GM1 to the patient to increaseGM1 levels in the affected tissues. This mechanism requires systemicadministration of large amounts of GM1 due to the poor ability of GM1 totransport into the brain, has short term benefits of immediate increaseof the levels, but suffers from some long term issues, includingpractical and safety concerns regarding sourcing GM1 from animal brains,the low bioavailability/penetrance of systemically administered GM1across the blood brain barrier, and no oral bioavailability requiringadministration by injection potentially multiple times per day orchronic intracerebral infusion. Therefore, chronic administration of GM1alone may be difficult and not ideal, based on currently availablemethods.

Therefore, a preferred embodiment relates to administering a genetherapy designed to increase expression of the human sialidase(neuraminidase) enzyme Neu3, which removes sialic acid residues fromcomplex polysialogangliosides converting them into GM1. Preliminarystudies have shown that administration of sialidase enzyme directly tothe brain in an animal model of PD has a neuroprotective effect similarto administration of GM1. This mechanism will increase GM1 levels in thetargeted tissue (substantia nigra and other brain tissues) and provide aneuroprotective effect similar to that obtained in clinical trialsperformed in which GM1 was administered to PD patients.

Sialidases, in particular, sialidase Neu3, hydrolyze sialic acidlinkages on gangliosides and can degrade complex gangliosides (and GD3)converting them into GM1. Thus, as described above, GD1b, GT1b, andGD1a, are partially converted to GM1 and the potentially apoptogenic GD3is partially degraded. Thus, application of Neu3 gene therapy willreshape the glycolipid composition of the cell, resulting in abeneficial variation in plasma membrane gangliosides and together withanti-apoptotic effects, will be neuroprotective. Accordingly, the genetherapy encoding for Neu3, provides for a mechanism to partially convertcomplex polysialogangliosides in the brain into GM1.

The sialidase can be administered via a gene therapy designed toincrease expression of GM1 wherein human compatible engineered AAVcontaining the Sialidase Neu3 sequence under an appropriate promoter isadministered. Administration of the AAV is preferentially administeredvia intracranial stereotaxic injection. In other suitable embodiments,administration of the AAV and the genes encoding for the sialidase Neu3gene may be administered via other mechanisms known to one of ordinaryskill in the art.

Actions of Sialidase

There are a number of different sialidases with somewhat differentactions. For example, sialidase C purified from Clostridium perfringens(CPS) preferentially cleaves a2-3 linkages, has comparatively littleactivity at a2-8 linkages and should result primarily in a conversion ofGD1a to GM1 (see below). Sialidase V purified from Vibrio cholerae (VCS)primarily cleaves a2-8 linkages and terminal a2-3 linkages and shouldresult in a net decrease in GD1a and b-series gangliosides GD1b and GT1band increased GM1. VCS should also degrade GD3. Therefore, theseincreases in endogenous GM1 levels (and decreases in potentiallydamaging GD3 levels) will be neuroprotective against dopamine neurondamage and death from a variety of causes including MPTP/MPP⁺-mediatedDA neuron toxicity as well as DA neuron toxicity mediated by a widevariety of other trigger mechanisms. Indeed, gangliosides GD3, GM3, GD1aand GD1b, but not GM1 or GM2, are good substrates for sialidase Neu3 forincreasing endogenous GM1 levels.

Indeed, FIGS. 1A and 1B depict diagrams of the ganglioside biosyntheticpathways, and depicts several mechanistic ways in which GM1 can beincreased through modification of the GM1 biosynthetic pathways. In FIG.1B, the underlined sialic acids are removed by the action of thesialidase, thus converting those gangliosides into GM1.

EXAMPLES

Human Embyonic stem cell (hES) culture: Human stem cells weredifferentiated into a dopaminergic (DAergic) phenotype using a wellcharacterized method (Iacovitti et al. 2007 as modified in Cai et al.,2013).

FIG. 2 depicts a brief diagram of the experimental overview. Depicted onthe top is the material administered and on the bottom is the number ofhours.

Rescue of DAergic hES cells: hES cells were plated onto 24 well plates.Cells were pretreated with sialidase, GM1 or standard media for 2 hrsand were then exposed to the mitochondrial toxin MPP⁺. Twenty-four hourslater, media containing toxin was removed and fresh media containing theappropriate concentration of rescue agent was added. Two days later,cells were fixed, processed for tyrosine hydroxylase (TH)immunocytochemistry and the number of TH immunopositive cells werecounted.

For in vivo studies, sialidases were prepared in artificial cerebralspinal fluid (aCSF) and loaded into Alzet osmotic pumps (model 2004).Brain infusion cannulae were attached to the pumps to allow forintracerebroventricular (ICV) administration of sialidases or vehicle.Brain infusion cannulae were placed in the third ventricle and osmoticpumps were implanted into C57B16 male mice (age 7-10 weeks). One weekafter surgery, sub-acute MPTP administration (2 injections 20 mg/kg s.c.for 5 days) was initiated. Mice were euthanized 2 weeks after the finalMPTP injection. In a separate group of animals, GM1 was administereddaily (30 mg/kg i.p.) beginning 24 hrs after the final MPTP injectionand continuing for two weeks.

Some animals receiving sialidase or aCSF alone were euthanized 12 dayspost-surgery to examine the effects of sialidase treatment on DA uptake,to examine if sialidase would interfere with DA uptake and inhibit theaccess of MPTP into DA neurons. Whole striatal homogenates were exposedto [³H] DA for 3 minutes in the presence or absence of mazindol toexamine the influence of sialidase treatment on DA uptake.

A the conclusion of the study, the striatum contralateral to the implantwas collected to analyze striatal catecholamine and metabolite levels byhigh performance liquid chromatography (HPLC) according to previouslypublished methods (Kidd and Schneider 2011). Briefly, samples weresonicated in perchloric acid and the soluble fraction was separatedusing MDTM mobile phase (11% acetonitrile, pH 3.0) on an ESA CoulochemIII HPLC system with electrochemical detection.

Sections were cut frozen at 30 μm on a sliding microtome. Every thirdsection through the rostro-caudal extent of the substantia nigra (SN)was processed for TH, the adjacent section was stained with cresylviolet and remaining sections were used for detection of gangliosides.All sections were washed in PBS, (endogenous peroxidase activity wasblocked using peroxide for IHC), blocked, and exposed to primaryantibody overnight. Sections were then exposed to biotinylatedsecondary, avidin biotin complex and developed for fluorescencemicroscopy.

Microbrightfield StereoInvestigator was used to estimate numbers of THimmunopositive and cresyl violet stained cells in the SN by unbiasedstereology, as described by us previously (Kidd and Schneider 2011).

FIGS. 3A and 3B depict the protection/rescue of human embryonic stemcells from MPP⁺ toxicity. A) The number of tyrosine hydroxylaseimmunopositive (TH⁺) cells was reduced as a result of MPP⁺ treatment.GM1 treatment prevented cell loss at the 5 and 10 μM doses of MPP⁺.*p<0.01 vs control, {circumflex over ( )}p<0.05 vs respective MPP⁺ only,**p<0.01 vs respective MPP⁺ only. B) Sialidase from Vibrio cholerae(VCS) was also able to protect/rescue cells from MPP⁺ toxicity.{circumflex over ( )}p<0.01 vs control; **p<0.01 vs respective MPP⁺only.

In mouse studies as depicted in FIG. 4, striatal uptake of tritiateddopamine (³H-DA) was examined. FIG. 6A depicts an experimental overviewof a dopamine uptake study. Striatal homogenates were incubated with³H-DA in the presence (non-specific) or absence (total) of the DA uptakeinhibitor mazindol (Maz). Mazindol effectively blocked total DA uptake;sialidase treatment did not, indicating that the sialidase does notinterfere with the mechanism of uptake of MPP⁺. FIG. 4 further showsthat specific uptake of DA in the striatum was not inhibited byintracerebral administration of sialidase to mice for 12 days.

FIG. 5 depicts immunofluorescent detection of gangliosides in mousestriatum. FIG. 5 depicts striatal sections showing an increase in GM1and a decrease in GT1b, GD1a, as a result of sialidase treatment.

FIG. 6B depicts a mouse study from days 0-26 wherein days 7-12 includethe animals being subjected to MPTP, the results of which are depictedin FIG. 7.

FIG. 7—Effects of sialidase on striatal dopamine levels by HPLC. MPTPtreatment resulted in significant decreases in striatal dopamine (DA).In mice that received sialidase infusion for 7 days prior to MPTP andthen for an additional 21 days, there was a significant sparing ofstriatal DA levels compared to animals that received control (aCSF)infusions. **p<0.01 vs aCSF-MPTP

FIG. 8—Effects of sialidase treatment on substantia nigra (SN) cells.Stereology was used to estimate the number of dopaminergic cells in thesubstantial nigra. Dopaminergic cells (TH⁺) with an identifiable nucleuswere counted at 100× magnification. Nissl cell counts (number of cresylviolet stained cells) were obtained using the same counting regions ofthe SN from an individual animal as used in adjacent TH⁺ sections (FIGS.8A, 8B: ***p<0.01 vs aCSF-MPTP; {circumflex over ( )}p<0.001 vsaCSF-MPTP). Sub-acute MPTP treatment resulted in significant decreasesin TH⁺ and Nissl-stained cell numbers. GM1 and sialidase treatment hadsimilar protective effects on SN dopamine neurons (FIGS. 8C (TH⁺ cells),8D (cresyl violet stained cells): **p<0.001 vs GM1; {circumflex over( )}p<0.0001 vs GM1; ˜p<0.001 vs GM1).

FIG. 9—AAV-hNeu3 expression in mouse substantia nigra (SN) 2 weeks afteradministration. An injection of AAV-hNeu3 (1.7×10¹⁰ GC in 1 μl) wasplaced just dorsal to the substantia nigra (SN) on one side of thebrain. Two weeks later, the animals were euthanized and the SN on eachside was dissected and frozen. RNA was extracted from the tissue samplesand the samples were run on RT-PCR to assess expression of the hNeu3transgene.

FIG. 10—Neuroprotection in an MPTP mouse PD model with AAV-hNeu3administration. AAV-hNeu3 was administered by injection (1.7×10¹⁰ GC in1 ul) just dorsal to the substantia nigra (SN) on one side of the brain.Two weeks later, MPTP was administered twice daily for 5 consecutivedays. Two weeks after the last MPTP injection, the animals wereeuthanized by transcardial perfusion and the brains sectioned andlabeled for visualization of green fluorescent protein (used as a tag onthe AAV to enable localization of the virus in tissue) and tyrosinehydroxylase (to label dopamine neurons). On the side of the brain whereAAV-hNeu3 was administered (ipsilateral), there were more dopamineneurons in the SN than compared to the opposite side of the brain thatdid not receive AAV-hNeu3 (contralateral).

Accordingly, the examples and studies identify that human embryonic stem(hES) cells, when differentiated into dopaminergic neurons, aresusceptible to MPP⁺ toxicity. However, there are mechanisms that existthat can mitigate the effects of MPP⁺ toxicity. Indeed, MPP⁺ toxicity inhES cells is mitigated by both GM1 and 0.005 U/mL VCS. Therefore,treatment with a sialidase, such as sialidase Neu3 that increases theamounts of GM1 expression in the striatum and the substantia nigra willprovide neuroprotective effects.

Furthermore, the experiments identify that sialidase treatment does notalter MPP⁺ uptake in vivo and that lack of MPP⁺ uptake is not themechanism for neuroprotection from sialidase administration. Conversely,sialidase treatment results in changes in ganglioside expression,resulting in an increase in GM1 expression, in both the striatum andsubstantia nigra. At least one concentration of sialidase enzyme had asparing effect on SN dopaminergic neurons. However, an effective amountof sialidase, administered to an animal, provided protective effects inmost circumstances. Indeed, some sialidase treatments were as effectiveas GM1 administration on sparing striatal dopamine levels and SNdopamine neurons.

These data show that increasing endogenous GM1 levels through the use ofsialidase enzymes can have significant protective/rescue effects ondopamine neurons, and at least in some instances, to the same extent asadministration of GM1 ganglioside. Since GM1 administration is limitedbased on the difficulty in sourcing the material and on lowbioavailability, other suitable materials are important for continuingto learn about and develop mechanisms to otherwise increase GM1 levelsin the brain. Therefore, sialidase treatment may be as effective assystemic GM1 administration in sparing striatal DA levels and SN neuronsand may be an alternative to systemic GM1 administration as a potentialPD disease modifying strategy.

An embodiment comprises a therapeutic treatment comprising a compositionof matter such as a pharmaceutical composition comprising a gene therapyvector expressing an exogeneous nucleic acid sequence comprising: (1) anucleic acid sequence encoding a functional Sialidase Neu3 enzyme; (2) apromoter operably linked to said nucleic acid sequence; and (3) whereinthe composition of matter is effective at increasing GM1 in the brain.

Indeed, appropriate methods of therapeutic treatment comprisingadministering the sialidase enzyme to a patient having aneurodegenerative disorder is suitable to prove protective effectsthrough increasing the levels of GM1 in the brain thereby providingprotective elements of GM1 and reducing or reversing the course ofneurodegenerative disease, such as PD, HD or other knownneurodegenerative diseases and conditions that are or may be affected byGM1. In certain embodiments, the sialidase enzyme is appropriatelydelivered through a composition comprising an AAV encoding for sialidaseNeu3. However, other suitable vectors are provided and described herein.

Indeed, it may be further advantageous to provide a therapeutictreatment comprising concomitant administration of GM1 and a genetherapy comprising an AAV encoding for a sialidase enzyme, Neu3, whereinthe direct administration of GM1 provides protective effects byincreasing GM1 directly and the gene therapy, through increase ofsialidase enzyme Neu3 administration can provide mechanistic changes inthe brain cells to protective effects through increasing the levels ofGM1 in the brain thereby providing protective elements of GM1 andreducing or reversing the course of neurodegenerative disease, such asPD, HD or other known neurodegenerative diseases and conditions that areor may be affected by GM1.

In appropriate settings, the compositions described herein andthroughout the embodiments may be advantageously used to treatneurodegenerative diseases or disorders, wherein use of the compositiongenerates an increase in GM1 in the brain. In certain embodiments, useof a composition comprising a viral vector for Sialidase Neu3 as amedicament for treatment of a neurodegenerative disease or disorder isappropriate.

A further therapeutic strategy may utilize the same pathway identifiedin FIG. 1 but approach the modification or increase of GM1 from adifferent aspect. In essence, the pathway for GM1 via the arrows depictsa natural flow, wherein GM2 is converted by B3Galt4 into GM1, and GM1then converted by St3Gal2 into GD1a, or GT1a. The sialiadase Neu3strategy increases GM1 levels by converting the downstream a-seriespolysialoganglioside GD1a and the b-series polysialogangliosides GD1band GT1b into GM1. However, a further strategy for increasing GM1 levelswould be to increase GM1 directly by correcting a possible defect orerror in the production of GM1 from GM2 due to decreased expression ofthe GM1 synthase enzyme, B3Galt4. This could be achieved through B3Galt4gene replacement.

Research suggests that in the substantia nigra in the PD brain, andparticularly in isolated dopamine neurons, there is a decreasedexpression of the mRNA for the enzyme B3Galt4(beta-1,3-galactosyltransferase 4)(AKA GM1 synthase), which contributesto decreased GM1 ganglioside levels in PD substantia nigra, which inturn, supports the degeneration of dopamine neurons. Unfortunately, nodrug has yet been developed that unequivocally has a positive effect ondisease progression. What is needed is a new treatment for PD that slowsthe degeneration of substantia nigra dopamine neurons and thus slows,arrests, or reverses the progression of the disease.

In addition to PD and HD, other neurodegenerative diseases such asAlzheimer's disease and ALS also suffer from similar GM1 reductionresulting in damage. Furthermore, several peripheral neuropathiesincluding, but not limited to: diabetic neuropathy, chemotherapy-inducedneuropathy such as “chemo-brain,” traumatic brain injury, acute spinalcord injury, stroke and retinopathies also may have similar patterns ofdamage resulting from low GM1 and would benefit from the treatmentsuggested herein. Alteration in GM1 biosynthesis may be a mechanismcommon to a wide variety of neurodegenerative disorders that mayincrease the vulnerability of neurons to degenerating in response to avariety of stressors.

In HD models and in HD fibroblasts, it has been shown that B3Galt4 mRNAis downregulated, resulting in decreased levels of GM1. It has also beenshown in HD models that intracranial administration of GM1 resulted innormalized motor function, a concomitant normalization of striataldopamine levels (critical to normal striatal function), andphosphorylation of mHtt at serine residues 13 and 16 that attenuatedmHtt toxicity. These data suggest that enhancing GM1 levels in HD may bedisease modifying.

Therefore, because each of HD and PD exhibit decreased GM1 gangliosidelevels and defects in B3Galt4 expression, mechanisms to support GM1levels or increase GM1 levels or correct the B3Galt4 defect will provideprotective effects in the brain.

Furthermore, because of the significant impediments to the therapeuticdelivery of GM1 in neurodegenerative disease, which include the possibleneed for chronic intra-cranial infusion or chronic systemic injection,practical and safety concerns regarding sourcing GM1 from animal brains,low bioavailability/penetrance of systemically administered GM1 acrossthe blood brain barrier, and no oral bioavailability requiringadministration by injection potentially multiple times per day or bycontinuous intracranial infusion, modification or increase of GM1 mustbe mediated through alternative means.

Accordingly, a more efficient way to increase GM1 levels will be bymodifying the cell's own ganglioside biosynthetic capacity utilizingviral or non-viral mediated gene therapy to increase expression ofB3Galt4, the enzyme responsible for GM1 synthesis and deficient in PDand HD. Therefore, a preferred embodiment of the invention isparticularly related to a method for treating neurodegenerative diseasesuch as PD or HD that is expected to have disease-modifying effects ofslowing the progression of the disease comprising increasing theexpression of B3Galt4 in the patient.

A preferred mechanism uses administration of a composition comprisingGM1 synthase cDNA in an expression vector to increase expression of theenzyme responsible for the direct biosynthesis of GM1. This willincrease endogenous levels of GM1 in substantia nigra neurons (in PD) orstriatal or cortical neurons (in HD) and leads to the same diseasemodifying effect as is seen with administration of GM1 ganglioside. Thismay be achieved alone or in combination with administration ofsubstances intended to increase levels of the precursor(s) for GM1.

Using adeno-associated virus (AAV) encoding these genes under a neuronspecific promoter, we will enhance endogenous GM1 expression following asingle intracranial administration. A preferred embodiment comprises acomposition of matter such as a pharmaceutical composition comprising agene therapy vector expressing an exogenous nucleic acid sequencecomprising: (1) a nucleic acid sequence encoding a functional B3Galt4enzyme; (2) a promoter operably linked to said nucleic acid sequence;and (3) wherein the composition of matter is effective at increasing GM1in the brain.

Accordingly, a proposed method comprises modifying the cell's ownganglioside biosynthetic capacity utilizing viral mediated gene therapyto increase expression of B3Galt4. Indeed, a human compatible engineeredadeno-associated virus (AAV) containing the B3Galt4 sequence under thecontrol of neuronal specific promoters can be administered to thepatient with neurodegenerative disease by intracranial stereotaxicinjunction. The AAV's containing the B3Galt4 sequence would then enhanceand/or normalize levels of GM1 in neurons, providing both therapeuticrelief and disease modifying effects. Furthermore, advances in AAVtechnologies now provide the potential for systemic administration ofAAV's with neuronal targeting in juveniles or adults, allowing fornon-surgical treatment of HD in newborns or in at-risk individuals whichmay further delay/inhibit the occurrence of the disease.

In further embodiments, other forms of gene therapy or other mechanismsof administration are suitable. For example, one of several humancompatible viral vectors are suitable for administering the genes ofinterest. This includes, but is not limited to such viral vectors aslentiviruses. Furthermore, it may be beneficial in certain embodimentsto use a lentiviral vector to transmit a B3Galt4 sequence, a sialidaseNeu3 sequence, or both to a patient.

In further embodiments non-viral vectors may be used for administeringthe genes of interest. These include use of liposomes, polymersomes,lipopolyplex, or bolaamphiphile nanovesicles (that can be targeted tospecific neuron subtypes). Therefore, certain targeted approaches can beutilized and formulated for specific intra cranial injection or throughIV.

In a further embodiment, in conjunction with B3Galt4 gene therapy, thelevels of GM1 can be modified through a further pathway wherein the GM1levels are not upregulated, but the breakdown of GM1 into othergangliosides can be reduced, therefore increasing the levels of GM1. Inone pathway, shRNA/siRNA is used to decrease expression of St3gal2 (ST3beta-galactoside alpha-2,3-sialyltransferase 2 “SIAT4B”). St3gal2 addssialic acid to the terminal galactose of Galβ1-3GalNAc-terminatedglycolipids, such as GM1 and GD1b, to synthesize GD1a and GT1b.Down-regulation of the gene for St3gal2 and inhibition of production ofthis enzyme results in decreased levels of GD1a and GT1b with aconcomitant increase in GM1. This has been shown to occur inSt3gal2-null mouse brain (Sturgill et al., 2012).

Therefore, a further proposed embodiment is directed to a composition toincrease endogenous GM1 ganglioside levels in the substantia nigra byreversing a defect in B3Galt4 through administration of B3Galt4 cDNA,alone, or in combination with shRNA/siRNA St3gal2, which will furtherincrease GM1 levels, wherein said composition of matter comprises a genetherapy vector expressing an exogeneous nucleic acid sequencecomprising: (1) a nucleic acid sequence encoding a functional B3Galt4enzyme; (2) a promoter operably linked to said nucleic acid sequence;(3) shRNA/siRNA St3gal2; and (4) wherein the composition of matter iseffective at increasing GM1 in the brain. Wherein the shRNA/siRNASt3gal2 may be operably linked to the therapeutic vector or provided asa separate medicament and administered concurrently with the therapeuticvector.

Therefore, a further proposed embodiment is directed to a method toincrease endogenous GM1 ganglioside levels in the substantia nigra byreversing a defect in B3Galt4 through administration of B3Galt4 cDNA,alone, or in combination with shRNA/siRNA St3gal2, which will furtherincrease GM1 levels.

In treatment of PD, principal administration is direct application ofthe gene therapy to the substantia nigra. While the treatment issomewhat invasive, PD patients currently receive brain surgery fromimplantation of deep brain stimulation electrodes. Furthermore, therehave been several gene therapy trials in PD including AAV2-GDNF andProSavin that required surgery. Other suitable routes of administrationare discussed throughout this application, wherein the appropriate doseand formulation can be modified by one of ordinary skill in the art.

In certain embodiments, use of a composition comprising a viral vectorcontaining cDNA for B3Galt4 as a medicament for treatment of aneurodegenerative disease or disorder is appropriate. Alternatively, useof a medicament comprising shRNA or siRNA st3gal2 is appropriate fortreatment of a neurodegenerative disease or disorder, or use of acombination of medicaments or therapeutics as described herein.

EXAMPLES

Tissue: Post-mortem tissue from PD patients and age matched controlswere obtained from the Human Brain and Spinal Fluid Research Center (LosAngeles, Calif.) or the NICHD Brain and Tissue Bank at the University ofMaryland.

Fresh frozen tissue was paraffin embedded and sectioned at 10 μm.Sections were briefly fixed in cold 80% acetone prior to neutral redstaining prior to laser capture microdissection (LCM). For LCM we usedthe PixCell II Laser Capture Microdissection instrument (ArcturusEngineering, Mountain View, Calif.). Only DA neurons in the SN with adefined nucleus and containing neuromelanin were sampled. RNA wasextracted and RNA amplicon libraries were generated for use with acustom AmpliSeq panel (focused on glycomics and trophic factors) andNext-Gen sequencing using the Ion Personal Genome Machine (PGM) System

Quantitative PCR Analysis:

RNA was extracted from fresh frozen samples using the Qiagen miRNeasykits according to manufacturer's instructions for use on the QIAcubesystem. Samples were converted to cDNA using the Qiagen Omniscript RTkit and oligo dT primer. Real-Time PCR was performed on cDNA using theRoche LightCycler 480 with Roche LightCycler 480 SYBR Green I Master. 25ng of cDNA was used per PCR reaction and all samples were analyzed intriplicate. The reaction conditions were as follows: B3galt4—95° C. for10 minutes and 45 cycles of 95° C. for 15 s and 60° C. for 1 minute. Toconfirm specificity of amplification the products were subjected to amelting curve analysis at the end of the final annealing period. Toanalyze the PCR data the ΔΔC_(t) method was used to calculatefold-regulation of glycosyltransferase mRNA expression relative to B2M(housekeeping gene) expression.

FIG. 11—GM1 treatment had an early symptomatic effect and a laterdisease modifying effect on PD. The mean (±SE) change from baseline inEarly-start (randomized to receive GM1 at start of study) andDelayed-start (randomized to receive placebo at start of study) studysubjects and in a standard-of-care comparison group (no interventions).The dashed vertical line at week 24 indicates the end of study Phase I.After that, all subjects received GM1. The dashed vertical line at week120 indicates the end of study Phase II. The horizontal dashed lineindicates baseline level. An increase of score indicates symptomworsening; a decrease in score indicates symptom improvement. *=p<0.0001Early-start vs. Delayed-start; {circumflex over ( )}=p<0.05 Early-startvs. Delayed-start

FIG. 12 depicts that Gangliosides are reduced in Parkinson's diseasesubstantia nigra. Thin layer chromatography data from the SN of 3 PDcases and 4 age-matched controls showing decreased expression of majorbrain gangliosides GM1, GD1a, GD1b, and GT1b. Therefore, modification ofthese major brain gangliosides via the mechanisms described herein,leads to neuroprotective effects for patients suffering from thesesneurodegenerative diseases.

FIGS. 13A and B depict that B3Galt4 gene and protein expression issignificantly altered in PD diseased brains. FIG. 13A depicts about atwo fold reduction of gene expression in PD brain SN versus age matchedcontrol. FIG. 13B further depicts the B3Galt4 Protein Expression in thesubstantia nigra in PD brain.

FIGS. 14A and B depict that B3Galt4 siRNA decreases GM1 expression inSK-N-SH Cells. Therefore, and as depicted in FIG. 14B, thedownregulation of B3Galt4 results in Enhancement of cell death whenB3Galt4 is downregulated. This again leads to the conclusion thatappropriate modulation of B3Galt4 can reduce the occurrence of celldeath or otherwise stabilize cell death to prevent or slow theprogression of disease.

FIGS. 15A and B depict the Downregulation of B3Galt4 in HD models and HDfibroblasts. Therefore, appropriate regulation of B3Galt4, as mediatedby the compositions and therapeutic methods or uses of the compositionsdescribed herein, can mediate the downregulation of B3Galt4 to reducethe neurodegenerative effects seen in the neurodegenerative diseases.

FIG. 16.—depicts side by side images of the contralateral andipsilateral brain images that identify the significant Neuroprotectiveeffect of AAV-B3 Galt4 in the MPTP mouse PD model. The B3Galt4 wasadministered on just one side of the brain, and wherein the side of thebrain where the B3Galt4 was administered, there were more neuronscompared to the opposite side of the brain that did not receive theB3Galt4.

GM1 levels appear to be reduced in the substantia nigra of Parkinson'spatients even in cells still expressing TH. Glycosyltransferase geneexpression is altered in the substantia nigra in Parkinson's patientsrelative to aged matched controls. Similar gene expression changes alsooccur in HD brain and in the brain in other neurodegenerative diseases.

Therefore, the present results suggest that there may be a dysregulationof ganglioside biosynthesis in PD (and HD) and that administration ofGM1 to PD patients (and in animal and cell models of HD) may provide aclinically important increase in GM1 levels in brain sufficient toovercome a PD (HD)-related GM1 deficit resulting in aneuroprotective/neurorestorative effect.

Because GM1 has proven to have neuroprotective effects in certainneurodegenerative diseases, increase of GM1 proves critical forprotecting and improving the life of those who suffer from suchdiseases. Described herein are several approaches to increasing GM1 inthe brain. Where all the strategies may work individually, so to may acombination therapy be relevant for appropriate treatment, and therapiesfor patients who suffer from these neurodegenerative disorders.Therefore, it is appropriate to generate a combination therapeuticcomposition that comprises both treatments to correct the B3Galt4 geneand also for increasing the Sialiadase Neu3. Such composition maycomprise a single viral or non-viral strategy that includes vectors foreach of the strategies, or, a composition may comprise separate vehiclesfor administering the two different strategies. Furthermore, combinedcomposition may further be administered with additional strategies asdescribed throughout this document.

Further methods may employ two separate compositions administeredconcurrently in a single medicament, or concurrently in time, with twoor more separate compositions or therapeutics administered separately,so that the effects are concurrently seen in the patient.

For example, an embodiment provides for gene therapy, including use ofviral vectors containing the sialidase Neu3 sequence of interest. Forexample, a particular viral vector includes the use of lentivirusescontaining the sialidase Neu3 sequence. In certain embodiments, the genetherapy can also include the B3Galt4 sequence alone, or together withsialidase Neu3. In certain embodiments, the gene therapy can alsoinclude the St3Gal2 sequence alone, or together with sialidase Neu3.Appropriate viral vectors include lentiviral vectors, retroviruses,adenoviruses, adeno-associated viruses; further more suitable non-viralvectors may also be implemented in certain embodiments.

Therefore, certain embodiments are directed to a concomitant therapy ofadministering a gene therapy encoding for the human sialidase enzymeNeu3 to increase GM1 levels and decrease GD3 levels in the brain,combined with a gene therapy designed to increase expression of GM1wherein human compatible engineered AAV containing B3Galt4 sequenceunder an appropriate promoter is administered via intracranialstereotaxic injection. The combination seeks to increase GM1 productionthrough effects on different GM1 biosynthetic pathways. The B3Galt4increases low levels of B3Galt4 in PD brain to enhance conversion of GM2into GM1 (and GD1b from GD2), whereas Neu3, when upregulated throughgene therapy, hydrolyzes sialic acid linkages on downstreampolysialogangliosides (including GD1b) to partially convert them to GM1to increase GM1 levels in the brain. The St3Gal2 increases low levels ofSt3Gal2 in PD brain to enhance conversion of GM1 into GD1a and GD1b intoGT1b, whereas Neu3, when upregulated through gene therapy, hydrolyzessialic acid linkages on GD1a and GT1b to partially convert them to GM1to increase GM1 levels in the brain.

1.-33. (canceled)
 34. A method of treating neurodegenerative disease ina subject in need thereof, the method comprising administering to saidsubject an effective amount of a vector encoding sialidase Neu3; whereinsaid vector modifies cells in said subject to increase production ofNeu3, thereby increasing the amount of GM1 in the brain of said subject.35. The method of claim 34, further comprising administering to saidsubject a vector encoding B3Galt4.
 36. The method of claim 34, furthercomprising administering to said subject a vector encoding B3Galt4,St3Gal2, or combinations thereof.
 37. The method of claim 36 whereinsaid vector encoding sialidase Neu3 and/or said vector encoding B3Galt4,St3Gal2, or combinations thereof is a viral vector selected from thegroup consisting of retrovirus, lentivirus, adenovirus, andadeno-associated virus (AAV) vector.
 38. The method of claim 36 whereinsaid vector encoding sialidase Neu3 and/or the vector encoding B3Galt4,St3Gal2, or combinations thereof is a non-viral vector.
 39. The methodof claim 34, further comprising concomitantly administering to saidsubject a dietary supplement with complex milk lipids (CMLs) having aconcentrated dietary source of GM3.
 40. The method of claim 34, furthercomprising concomitantly administering shRNA/siRNA St3gal2 to increaseGM1 levels.
 41. The method of claim 34, wherein the neurodegenerativedisease is Parkinson's disease or Huntington's disease.